Peroxidative damage of mitochondrial respiration is substrate-dependent

Summary The concentration-dependence of tert-butyl hydroperoxide (BHP) inhibitory effect on oxygen consumption in isolated rat liver mitochondria was measured in the presence of various respiratory substrates. Strong inhibitory effect at low concentrations of BHP (15-30 µM) was found for oxoglutarate and palmitoyl carnitine oxidation. Pyruvate and glutamate oxidation was inhibited at higher concentrations of BHP (100-200 µM). Succinate oxidation was not affected even at 3.3 mM BHP. Determination of mitochondrial membrane potential has shown that in the presence of NADH-dependent substrates the membrane potential was dissipated by BHP but was completely restored after addition of succinate. Our data thus indicate that beside peroxidative damage of complex I also various mitochondrial NADH-dependent dehydrogenases are inhibited, but to a different extent and with different kinetics. Our data also show that succinate could be an important nutritional substrate protecting hepatocytes during peroxidative damage

Tetraphenylphosphonium-Selective Electrode as a Tool for Evaluating Mitochondrial Permeability Transition Pore Function in Isolated Rat Hepatocytes

Abstract. The changes in mitochondrial membrane potential (∆ψ m ) were used as an indicator for evaluating the mitochondrial permeability transition pore (MPTP) function. We found that in situ mitochondria in digitonin-permeabilized hepatocytes were coupled and responded to the addition of substrates, inhibitors and uncouplers. Ca 2+ -induced ∆ψ m dissipation was caused by MPTP opening because this process was inhibited by cyclosporin A. MPTP opening was enhanced by the pro-oxidant tert-butyl hydroperoxide

Tert-Butyl Hydroperoxide Selectively Inhibits Mitochondrial Respiratory-Chain Enzymes in Isolated Rat Hepatocytes

Summary Sensitivity of various mitochondrial enzymes to oxidative damage was tested on isolated rat liver hepatocytes permeabilized by digitonin. In permeabilized hepatocytes normal respiratory control values were obtained and mitochondrial membranes remained intact. Respiratory rates of NADH-dependent (glutamate+malate, palmitylcarnitine + malate) and flavoprotein-dependent (succinate) substrates were determined in hepatocytes exposed for 5 min to 0.5-3 mM tert-butyl hydroperoxide before addition of digitonin. Our data showed that oxidation of NADH-dependent substrates is much more sensitive to oxidative stress than oxidation of flavoprotein-dependent ones, evidently due to the modification of iron-sulfur clusters or SH groups in the NADH dehydrogenase enzyme complex (Complex I)

Antioxidant Vitamin Levels and Glutathione Peroxidase Activity During Ischemia/Reperfusion in Myocardial Infarction

Summary The consequences of increased oxidative stress, measured as the level of malondialdehyde (MDA) during ischemia/reperfusion, were studied in 48 patients in the acute phase of myocardial infarction (AMI) and a control group (21 blood donors). The serum levels of α-tocopherol and β-carotene were followed. Immediately after the treatment onset the level of α-tocopherol started to decrease, reaching a plateau after 24 h. The consumption of β-carotene was delayed by 90 min. Steady decline was detected during the whole time interval studied (48 h). Glutathione peroxidase (GPx) activity, as a representative of antioxidant enzymes, was estimated in whole blood. The influx of oxygenated blood was accompanied by a stimulation of GPx activity, which reached its maximum at the time of completed reperfusion. When comparing the AMI patients with the control group, the levels of MDA were found significantly increased, which indicates that oxidative stress is already increased during ischemia. Lower antioxidant levels found in the patients might either already be the result of vitamin consumption during ischemia or be a manifestation of their susceptibility to AMI. Monitored consumption of α-tocopherol and β-carotene during reperfusion indicated that in the case of patients, whose level of antioxidant vitamins is below the threshold limit, a further substantial decrease of antioxidant vitamins during reperfusion could enhance the oxidative damage of the myocardium